Received: February 8, 2010 Accepted: June 30, 2010
Conflict of Interest Statement: The authors state that there are no financial and personal conflicts of interest that could have inappropriately influenced their work.
Copyright: © 2010 Bezerra Júnior et al.; licensee EDIPUCRS. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 Unported License.
Evaluation of organic and inorganic
compounds in the saliva of patients with
chronic periodontal disease
Avaliação de compostos orgânicos e inorgânicos na saliva
de pacientes com doença periodontal crônica
Arnaud Alves Bezerra Júnior a
Débora Pallos b
José Roberto Cortelli b
Cintia Helena Coury Saraceni c
Celso Silva Queiroz c
aResearch Fellowship, Department of Dentistry,
University of Taubaté, Taubaté, SP, Brazil
bDepartment of Dentistry, University of Taubaté,
Taubaté, SP, Brazil
cDepartment of Dentistry, Paulista University, São
Paulo, SP, Brazil
Correspondence:
Celso Silva Queiroz
Rua Dr. Bacelar, 1212 – 4º andar Vila Clementino
São Paulo, SP – Brasil 04026-002
E-mail: celsoq@yahoo.com
Abstract
Purpose: The aim of this study was to evaluate the influence of periodontal disease on the biochemical parameters of the saliva, including salivary flow rate, pH, total protein, alkaline phosphatase activity and urea concentration, in individuals with chronic periodontitis. Methods: Forty subjects were allocated into two groups: twenty subjects without periodontal disease (control group) and twenty subjects with chronic periodontitis (test group). Plaque index (PI), gingival index (GI), probing depth (PD) and clinical attachment level (CAL) were obtained by a previously trained and calibrated clinical examiner. Saliva from both groups was collected for analysis of its biochemical parameters. The Student’s t-test and correlation coefficient (Pearson) were used at a 5% level of significance.
Results: The results showed alterations in the alkaline phosphatase activity, urea concentration and total protein in the subjects of the test group compared to the control group (P<0.05). However, there was no correlation between the clinical periodontal parameters and the salivary parameters (P>0.05).
Conclusion: The results suggest that chronic periodontitis can affect the composition of the saliva and that analysis of the salivary parameters can be useful as an additional exam for the diagnosis of periodontal disease.
Key words: Dental plaque; periodontitis; saliva; alkaline phosphatase; urea
Resumo
Objetivo: O objetivo deste estudo foi avaliar a influência da doença periodontal sobre os parâmetros bioquímicos da saliva, incluindo taxa de fluxo salivar, pH, proteína total, atividade de fosfatase alcalina e a concentração de uréia, em indivíduos com periodontite crônica. Metodologia: Quarenta indivíduos foram alocados em dois grupos: vinte indivíduos sem doença periodontal (grupo controle) e vinte indivíduos com periodontite crônica (grupo teste). Índice de placa (IP), índice gengival (IG), profundidade de sondagem (PD) e nível de inserção clínica (CAL) foram obtidos anteriormente por um examinador treinado e calibrado clinicamente. Saliva de ambos os grupos foram coletadas para análise de seus parâmetros bioquímicos. O teste T de Student e coeficiente de correlação (Pearson) foram utilizados em um nível 5% de significância.
Resultados: Os resultados mostraram alterações na atividade da fosfatase alcalina, uréia e proteína total nos indivíduos do grupo teste em comparação ao grupo controle (P<0,05). No entanto, não houve correlação entre os parâmetros clínicos periodontais e os parâmetros salivares (P>0,05).
Conclusão: Os resultados sugerem que a periodontite crônica pode afetar a composição da saliva e que a análise dos parâmetros salivares pode ser útil como um exame complementar para o diagnóstico da doença periodontal.
Introduction
Chronic periodontitis is an infectious disease that results in inlammation of the protective tissues of the teeth and the progressive loss of conjunctive tissue and alveolar bone. It is the most frequently found periodontal patho- logy, and although its occurrence normally involves adult individuals, periodontitis can appear at any age and even affect dentitions (1).
The diagnosis of periodontal disease is accomplished through clinical periodontal parameters including plaque index, gingival index, probing depth and clinical attachment level; however, the analysis of saliva can contribute to the diagnosis and prognosis of the disease (2). The organic and inorganic composition of saliva can supply information about alterations in enzymatic activity in response to periodontal inlammation (3).
Alkaline phosphatase (ALP) is a calcium- and phosphate-binding protein and a phosphor-hydrolytic enzyme. ALP is considered to be an important indicator of bone formation and is a phenotypic marker for osteoblast cells (4). ALP was detected in the parotid, submandibular and minor salivary glands, as well as in desquamated epithelial cells, leucocytes and bacteria from dental plaque. The presence of the ALP in the saliva and in the gingival crevicular luid is usually indicative of inlammation and/or destruction of the periodontal tissues. The level of ALP is positively correlated with the severity of the periodontal disease (5).
Among the several salivary components, urea is an organic compound that represents the inal product of the human catabolism of proteins. The hydrolysis of urea by bacterial urease enzymes generates ammonia and CO2, and is
considered a major pathway for alkali production in the oral cavity (6). In addition, ammonia is potentially cytotoxic for the periodontal tissues (6). Ammonia can increase the permeability of the sulcular epithelium to other antigenic and toxic substances, thereby playing a fundamental role in the initiation of gingivitis (7).
The volume of saliva in the oral cavity depends on several factors, such as stimulation, circadian rhythm, diet, age, drugs and the hydrogen (H+) ion concentration (8). Meanwhile,
these factors can also be altered due to pathological conditions, such as periodontal disease (8).
The purpose of this study was to analyze the salivary parameters (low rate, pH, total protein, alkaline phosphatase and urea) of individuals with chronic periodontitis compared to healthy individuals. Therefore, the null hypothesis of the present study was that there are no changes of the salivary parameters in individuals with periodontal disease.
Methodology
Subjects
This study was previously submitted and approved by the Ethics Committee in Research of the University of Taubaté (protocol number: 252/08), and voluntary informed consent was signed by the participants. A pilot study was conducted
in order to determine the size of sample, and ive individuals with periodontal disease were compared to ive individuals without periodontal disease. The data obtained from the salivary and clinical parameters were statistically analyzed (Student test, α=95% and power of 0.8) by the Software BioEstat 4.0. In accordance to the previous sample size calculation, forty individuals were allocated into two groups: a control group (twenty subjects without periodontal disease) and a test group (twenty subjects diagnosed with chronic periodontitis).
All patients met the following inclusion criteria: had more than 20 teeth, they had not taken antibiotics or anti-inlammatory drugs in the previous 6 months, had no immunodeiciency, were healthy in general terms. Patients who were smokers or who used anxiolytics, hypnotics, antidepressants, antihistaminic, diuretics or narcotics were excluded from this study.
The volunteers in the control group showed no clinical evidence of chronic periodontitis or gingivitis, probing depth showed no bleeding (1).
Volunteers in the test group had two periodontal pockets in each quadrant with probing depth of 5 mm in one or more sites and presence of clinical atachment level 6 mm in two or more teeth (9).
Evaluation of the clinical parameters
The plaque index (PI) (10), gingival index (GI) (11), probing depth (PD) and clinical attachment level (CAL) were measured by a clinical examiner who was previously trained and calibrated. The PD was accomplished at six points per tooth.
Collection and analysis of salivary parameters
The saliva samples were collected from the patients and controls between 9:00 and 10:00 in the morning, with no oral intake of food or drink. The patients were in a seated position, with the head inclined forwards. The saliva was collected in sterilized and refrigerated tubes during 5 minutes period and immediately the saliva samples were centrifuged at 3000 rpm for 5 minutes.
The unstimulated salivary low was deined as the total volume of saliva produced per unit time (mL/min) (12). The pH values of the saliva were immediately measured using a pH meter probe (PHB-Master model, Gulton, Brazil). Afterwards, 1000 µL was stored at -20oC until they were
processed.
The protein concentration of the supernatant luid was determined using a biuret reaction (13) that incorporates the use of Folin – Ciocalteau phenol reagent for enhanced color development (14). ALP activity was measured by colorimetric method, using monophosphate of timolftalina as the substrate (15). The saliva samples were incubated at 37°C for ten minutes, and the total activity of alkaline phosphatase was provided.
pentoxide in acid solution (sulfuric acid) like reagent. The standard solution containing 1.0 g urea/100 mL was used to prepare the standard curves. Each saliva sample was assayed in triplicate identical reactions.
The study variables were statistically analyzed at a 5% level of signiicance using the Student’s t-test and Pearson correlation coeficient.
Results
The demographic data for the groups are presented in Table 1. The mean values and standard deviations of the salivary parameters for the control and test groups are shown in Table 2. Signiicant differences were observed between control and test groups for all studied variables except salivary low rate.
The correlation of the salivary parameters is shown in Table 3. There was a positive, statistically signiicant corre-lation between the pH value and urea concentration, but the other correlations were not statistically signiicant. The correlation between the clinical and salivary parameters showed a low correlation between the variables of the study, without statistical signiicance (P>0.05) (Table 4).
Test group Control group
n 20 20
gender 4 m 16 f 8 m 12 f
age 42.27±8.07 28.3±6.95
PI 1.85±0.30 0.40±0.49
GI 1.82±0.34 0.25±0.47
PD 6.44±0.98 1.58±0.23
CAL 3.75±1.16 0.67±0.64
Table 1. Description of the number of individuals (n), age,
frequency of male (m) and female gender (f), plaque index (PI), gingival index (GI), probing depth (PD) (in millimeters) and clinical attachment loss (CAL) (in millimeters).
Table 3. Correlation among the salivary
parameters: flow rate (FR), pH, total protein, alkaline phosphatase (ALP) and urea concentration.
Table 4. Correlation among the salivary
parameters: flow rate (FR), pH, total protein, alkaline phosphatase (ALP) and urea concentration; and the clinical periodontal parameters: plaque index (PI), gingival index (GI), probing depth (PD) and clinical attachment loss (CAL).
Table 2. Averages and standard deviations of the salivary
parameters: salivary flow (SF), pH, total protein, alkaline phosphatase (ALP) and urea concentration in the control and test groups.
Test group Control group SF (mL/min) ns 2.33±1.11 2.88±1.07
pH** 7.71±0.43 6.91±0.18
Protein (mg/dL) ** 599.15±51.86 618.09±17.52
ALP (U/L) ** 16.79±1.72 10.63±1.48
Urea (mg/dL) ** 117.99±12.52 52.67±7.27
** statistical significance – (P<0.05). ns = not significant – (P>0.05).
Salivary parameters Salivary parameters
FR PH Protein ALP Urea
FR 1.000 ---- ---- ----
----pH 0.35 ns 1.000 ---- ----
----Protein -0.35 ns -0.06 ns 1.000 ----
----ALP 0.40 ns 0.08 ns -0.23 ns 1.000
----Urea 0.23 ns 0.50** -0.31 ns 0.16 ns 1.000
** statistical significance – (P<0.05). ns = not significant – (P>0.05).
Clinical periodontal parameters
Salivary parameters
FR pH Protein ALP Urea
PI 0.20 ns 0.15 ns -0.40 ns -0.23 ns 0.05 ns
GI -0.17 ns 0.13 ns 0.45 ns 0.05 ns -0.01 ns
PD -0.22 ns -0.15 nsM 0.43 ns -0.16 ns -0.07 ns
CAL -0.46 ns -0.007 ns -0.13 ns 0.37 ns -0.14 ns
ns = not significant (P>0.05).
Discussion
The pH value of the saliva was higher in the group with periodontal disease. This increase can be due to the urea concentration, which is diffused from saliva to the dental plaque. The urea is metabolized quickly by bacterial urease enzymes, producing ammonia and carbonic gas and causing an elevation in the pH of the saliva (17).
The caries index was not evaluated in the present study. However, in a recent study by Shu et al. (18), a larger urea level was veriied in the dental plaque of caries-free individuals, suggesting that enzymatic activity can increase the pH and decrease the development of caries.
The total protein concentration was smaller in the test group’s saliva. This result can be connected to the increase in proteolysis activities that occur in periodontal pathological conditions (19).
an increase of the ALP activity in patients with moderate and severe periodontitis.
The urea concentration in the saliva was higher in the test group, and this result corresponds with the study of Nomura et al. (21). This increase in urea is associated with bacterial urease activity, especially from gram-negative anaerobic bacteria, and the consequent formation of ammonia. Ammonia can be cytotoxic for the periodontal tissues (6). The correlation analyses between the clinical and salivary parameters were not signiicant. This result showed that the clinical parameters of individuals with periodontal disease as gingival bleeding, plaque, periodontal pocket does not necessarily indicate changes in pH, total protein, alkaline phosphatase activity and concentration of urea. Perhaps, if the salivary parameters evaluated in this study were related to inlammatory agents (interleukins, growth factors) could be correlated with clinical parameters. However, there was a positive correlation between urea and pH levels (r=0.50 and p=0.035). This result can be explained by the catalytic activity of the urease enzyme and the consequent formation of ammonia, which can increase the pH of saliva (16). The chronic periodontitis usually affects adults, and can be identiied by some clinical features themselves as the presence of calculus and plaque in an amount consistent with periodontal destruction of the individual, which is of low intensity and installed a long period (1). However, additional tests such as X-rays and tomography are useful in the diagnosis of periodontal disease.
The salivary parameters can also contribute for the diagnosis of periodontal disease, since saliva is a mixture
of oral luid, its composition may relect the composition of genvival luid crevicular. Although this study has only assessed ive salivary parameters, it is known that the presence of microorganisms existing in saliva may have a direct relationship with periodontal disease (1). There is a close association between juvenile periodontitis and
Actinobacillus actinomycetemcomitans, there is evidence
between periodontal disease and the development of several systemic diseases, among these, cardiovascular diseases (22), arthritis (23), pneumonia (24,25) and diabetes (26). The reason for the interest in establishing the parameters salivary how an additional tool for diagnosis of periodontal disease, is being able to distinguish a variety of components that might be useful biomarkers to detect and monitor changes in oral health. The noninvasive nature of saliva testing has been increasingly attractive to use as alternatives to blood.
Conclusions
The results suggest that chronic periodontitis can affect the composition of the saliva and that analysis of the salivary parameters can be useful as an additional exam for the diagnosis of periodontal disease.
Acknowledgments
The authors express their gratitude to Mr. José Antônio Domingos from the Laboratory of Biochemistry, UNESP, for his technical assistance.
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